JP5171668B2 - Method for correcting substrate misalignment - Google Patents

Description

  The present invention relates to an apparatus for performing fluorescence observation using evanescent light.

  When observing a biological substance such as DNA or protein, a method of observing the generated fluorescence by labeling with a fluorescent dye and irradiating excitation light such as a laser is common. In recent years, there is an observation method using evanescent light as a technique that enables fluorescence measurement at a single molecule level (Non-Patent Document 1). When light having a certain angle or more enters from a medium having a high refractive index toward a medium having a low refractive index, the incident light does not propagate to the medium having a low refractive index and total reflection occurs. At this time, a phenomenon occurs in which light slightly oozes out on the low refractive index medium side surface of the boundary surface. This leaking light is called evanescent light. The intensity of the evanescent light attenuates exponentially as the distance from the refractive index boundary plane increases, and the excitation light intensity becomes 1 / e at a distance of about 150 nm from the refractive index boundary plane. For this reason, by fixing the observation sample to the boundary surface, it becomes possible to irradiate only the sample near the boundary surface with excitation light, suppress background light originating from free phosphors and Raman scattering of water, etc. A contrast image can be obtained.

  Furthermore, DNA sequencing using the aforementioned single-molecule fluorescence detection technique has been proposed (Non-Patent Document 2). Sample DNA fragments to be analyzed are randomly captured on the surface of the substrate one molecule at a time, extended by approximately one base, and measured by fluorescence detection using the aforementioned evanescent light. The base sequence is determined from the measurement result. Specifically, a single target DNA molecule is immobilized on the solution layer side on the refractive index boundary plane by utilizing biotin-avidin protein binding. The DNA polymerase can be incorporated into the template DNA as a substrate for the DNA polymerase and the DNA chain elongation reaction can be stopped by the presence of a protecting group, and the DNA polymerase can be detected using four types of dNTP derivatives (MdNTP) having a detectable label. The step of performing the reaction, the step of detecting the incorporated MdNTP by fluorescence or the like, and the step of returning the MdNTP to a state where it can be extended are defined as one cycle. By repeating this, the base sequence of the sample DNA is determined.

  A so-called autofocus function for correcting focus is used in various devices today. For example, the microscope described in Patent Document 1 has an autofocus function.

JP 2002-341234 A

Nature 1995, Vol. 374, pp. 555-559. PNAS 2003, Vol. 100, pp. 3960-3964.

  As a result of intensive studies on the apparatus for performing fluorescence observation using evanescent light, the present inventor has obtained the following knowledge.

  While evanescent light irradiation has the advantage of being able to observe a weak signal with a low background, it has the demerit that strict focus adjustment must be performed. A lens used for single molecule observation generally has a high magnification and a high NA. Therefore, the depth of focus is shallow, and the focus is shifted by a slight change in the thickness of the substrate. Each time the observer moves the field of view, it is necessary to check whether there is a focus shift and to correct as necessary.

  However, autofocus represented by Patent Document 1 is insufficient for single-molecule fluorescence observation. In fluorescence observation by evanescent irradiation, the tilt of the substrate also affects the observation result. This is because the incident angle of the excitation light changes due to the inclination of the substrate surface, and the intensity distribution of the evanescent light changes. Although the surface of the substrate is smooth at first glance, since the unevenness exists locally, the incident angle changes for each irradiation region. As a result, the excitation light intensity is not constant, and the observation results vary. In order to obtain a stable result, it is necessary to keep the relative positional relationship between the sample surface and the objective lens constant (preferably parallel). That is, when performing single molecule fluorescence observation, it is necessary to adjust the tilt in addition to the substrate Z-axis direction.

  An object of the present invention is to provide a fluorescence observation method and apparatus having good operability, high sensitivity, and high reliability.

  The present invention irradiates an observation substrate for performing fluorescence observation using evanescent light with light of a specific wavelength, adjusts the focus by observing the fluorescence and scattered light generated thereby, and coordinates of the irradiated light And adjusting the tilt of the substrate based on the result.

  According to the present invention, manual adjustment operation is not necessary in fluorescence observation using evanescent light. It is also possible to keep the observation substrate at a constant angle, and the incident angle of the excitation light is constant, so that the penetration depth of the evanescent light does not change between the observation fields, improving the stability and reliability of fluorescence observation. . In addition, the irradiation area of the evanescent light can always exist at the center of the field of view, and there is no fear of observation loss, so that the throughput can be improved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. FIG. 3 is an explanatory diagram of a reaction substrate in Example 1. FIG. 3 is a flow chart of observation in Example 1. FIG. 6 is an explanatory diagram of an apparatus according to the second embodiment. FIG. 6 is an explanatory diagram of an apparatus according to a third embodiment. FIG. 10 is a flow chart of observation in Example 3. FIG. 6 is an explanatory diagram of an apparatus according to a fourth embodiment. FIG. 10 is a flow chart of observation in Example 4.

  The embodiment is a method for detecting and correcting a change or inclination in the Z-axis direction of the observation substrate caused by the movement of the stage in the apparatus for performing fluorescence observation using evanescent light, and keeping the observation conditions for each field of view constant. About. This method irradiates the observation substrate with light of a specific wavelength, adjusts the focus by observing the fluorescence or scattered light generated thereby, obtains the coordinates of the irradiated light, and determines the substrate based on the result. Adjusting the inclination of the.

  Further, in the embodiment, adjustment light is radiated on the substrate surface, and fluorescence and reflected light are detected, thereby correcting variations in conditions between observation fields. In this method, a step of irradiating the observation visual field with adjustment light, a step of detecting the resulting fluorescence, a step of adjusting the focus based on the detection result, and a step of detecting the reflected adjustment light And a step of correcting the tilt of the substrate based on the detection result.

  Further, in the embodiment, the condition variation between the observation fields is corrected by irradiating the substrate surface with the adjustment light and detecting the scattered light and the reflected light. In this method, a step of irradiating the observation field with adjustment light, a step of detecting the resulting scattered light, a step of adjusting the focus based on the detection result, and a step of detecting the reflected excitation light And correcting the tilt of the substrate based on the detection result.

  In the embodiment, the substrate surface is irradiated with adjustment light, and the scattered light and the shape of the irradiation spot are analyzed to correct the variation in conditions between the observation fields. In this method, the step of irradiating the observation field with excitation light, the step of detecting the resulting scattered light, the step of adjusting the focus based on the detection result, and the irradiation spot shape of the irradiated adjustment light A step of detecting, a step of calculating an aspect ratio of the spot from the detection result, and a step of correcting the tilt of the substrate based on the calculated result.

  In the embodiment, based on the detection result, the substrate on which the measurement sample is arranged, the light source that irradiates the substrate, the detector that detects the light emitted from the substrate, the drive mechanism that adjusts the position of the substrate, and the like. Disclosed is a total reflection fluorescence observation apparatus having an arithmetic unit that analyzes a relative position of a substrate in the apparatus and controls a driving mechanism.

  In the embodiment, the fluorescence observation apparatus using evanescent light is a method for correcting the positional deviation of the substrate accompanying the stage movement, and the substrate surface is irradiated with adjustment light, and at least one fluorescence generated by the irradiation is emitted. The light for adjustment reflected on the surface of the substrate is detected by at least one optical sensor, and the relative position of the substrate in the device is analyzed by the arithmetic unit based on the detection result. A method for adjusting the position of a substrate based thereon is disclosed.

  In addition, in the embodiment, in the fluorescence observation apparatus using evanescent light, a method of correcting the positional deviation of the substrate accompanying the stage movement, the substrate surface is irradiated with adjustment light, and at least fluorescence generated by the irradiation is emitted. An aspect ratio is calculated from the shape of the irradiation spot detected on a single optical sensor and the surface of the substrate is calculated by an arithmetic unit, and the relative position of the substrate in the apparatus is analyzed by the arithmetic unit based on the detection result and the aspect ratio. A method for adjusting the position of the substrate based on the analysis result is disclosed.

  Moreover, in an Example, it discloses that the said board | substrate is formed with the member which permeate | transmits light.

  Moreover, in an Example, it discloses that the said measurement sample exists on the structure which exists in the surface of a board | substrate.

  Moreover, in an Example, it discloses that the said structure is the microparticles | fine-particles or protrusion which consist of a metal element or a compound, and a measurement sample exists in the surface of the said structure.

  Moreover, in an Example, it discloses that the said structure is a micropore provided in the metal thin film of the surface of a board | substrate, and a measurement sample exists in the inside of a micropore.

  In addition, the Examples disclose that the wavelength of the light emission is larger than the wavelength of the excitation light.

  Moreover, in an Example, it discloses that the said drive mechanism is provided in the stage holding a board | substrate.

  In the embodiment, it is disclosed that the drive mechanism adjusts the height direction of the stage and the rotation direction with respect to the XY axes.

  The above and other novel features and effects will be described below with reference to the drawings. The drawings are used exclusively for understanding the invention and do not reduce the scope of rights. Moreover, each Example can be combined suitably.

  FIG. 1 shows a configuration diagram of a DNA base sequence analyzer having an adjustment function in the present embodiment. The apparatus has an inverted microscope-like configuration and measures fluorescent molecules captured by the reaction substrate 1 by fluorescence detection. It is also possible to adopt an upright apparatus configuration. Moreover, when based on the single molecule fluorescence detection method, the measurement is performed in a clean room-like environment through a HEPA filter.

  The laser beam 2 a from the excitation laser device 2 passes through the λ / 4 wavelength plate 3 to become circularly polarized light, enters the prism 6 through the lens 4 and the deflection element 5, and is irradiated from the back side of the reaction substrate 1. The The prism 6 and the reaction substrate 1 are in contact with each other via matching oil, and the laser light 2a is incident on the reaction substrate 1 without being reflected at the interface. The materials of the prism, the matching oil, and the reaction substrate are desirably equal in refractive index in consideration of light reflection loss. For example, the prism and the reaction substrate are made of synthetic quartz, and the matching oil is non-fluorescent glycerin. The laser beam 2a is totally reflected on the surface of the reaction substrate 1 to generate evanescent light. The incident angle θ at this time is obtained by the following equation from the refractive index of the reaction substrate 1 and the refractive index of the medium existing on the substrate surface. For example, when the reaction substrate 1 is synthetic quartz and the medium is water, the incident angle θ is about 66 degrees.

(Formula) θ = arcsin (n2 / n1)
n1: Refractive index of substrate, n2: Refractive index of medium, n1> n2

  The reaction substrate 1 is fixed to a transmission type XY automatic stage 7. The stage 7 is provided with a Z-axis drive mechanism 8 for adjusting the height of the reaction substrate 1 and an α / β-axis drive mechanism 9 for adjusting the inclination. The laser beam 2a is incident so that evanescent light is generated at the rotation center of the α / β-axis drive mechanism 9. The emitted light 10 on the reaction substrate 1 is collected by the objective lens 11, the wavelength component is selected by the filter unit 12, and detected by the two-dimensional sensor camera 14 through the imaging lens 13. The filter unit 12 holds a plurality of filters corresponding to the wavelength to be detected. For example, when observing dNTP to which four types of fluorescent dyes are added, the corresponding four types of phosphor filters are switched by the filter unit 12 to detect a fluorescent image. The two-dimensional sensor camera 14 is, for example, an EM-CCD camera having a pixel size of 16 × 16 μm and a number of pixels of 512 × 512 pixels. In addition to general cooling CCD cameras, various imaging cameras such as C-MOS area sensors can be used. The two-dimensional sensor camera 14 is preferably a cooling type, and by setting the temperature to about −20 ° C. or less, dark noise of the sensor can be reduced and measurement accuracy can be improved. Each component is installed so that the rotation center of the XY stage 7 is the center of the observation field.

  The adjustment light source 18 is applied to the surface of the reaction substrate 1 from the side opposite to the laser device 2 that is an observation light source. The adjustment light source 18 preferably has a wavelength different from that of the laser device 2 so that the measurement sample is not quenched by irradiation. For example, an LD having a wavelength of 694 nm can be used as the adjustment light source 18. The LD light 16 is applied to the reaction substrate 1 through the half mirror 17. The light reflected by the surface of the reaction substrate 1 passes through the half mirror 17 and reaches the two-dimensional sensor camera 14. At this time, since the optical axis on which the LD light 16 is incident on the reaction substrate 1 is coaxial with a straight line connecting the center of the XY stage 7 and the center of the two-dimensional sensor camera 14, the LD light 16 is irradiated to the center of the observation field. .

  Various reagents and buffers are stored in the reagent storage unit 15 and are sent to the reaction substrate 1 through the liquid supply tube 32 via the dispensing unit 31. The reagent after the reaction accumulates in the waste liquid container 19 through the waste liquid tube 33. The operation of the dispensing unit 31 is controlled by the control PC 20 together with the Z-axis drive mechanism 8, the α / β-axis drive mechanism 9, the filter unit 12, and the two-dimensional sensor camera 14. The acquired observation data can be confirmed on the monitor 21.

  FIG. 2 is an enlarged view of the surface of the reaction substrate 1. The reaction substrate 1 includes a transparent member 22 and a structure 23. As the transparent member 22, for example, synthetic quartz can be used. The structure 23 is, for example, Au. A measurement sample 24 is fixed to the surface of the structure 23. For example, the end of the measurement sample 24 is thiolated, and is fixed to the structure 23 via an Au—SH covalent bond. The structures 23 may be arranged randomly on the transparent member 22, but are preferably arranged regularly in consideration of efficiency during observation. For the region other than the structure 23 of the transparent member 22, for example, a metal thin film for shielding background light during observation may be disposed.

FIG. 3 is a flowchart of fluorescence observation in the present example. A specific method is performed according to the following procedure. After fixing the reaction substrate 1 to the XY stage 7, the adjustment LD light 16 is irradiated to the first visual field on the surface of the reaction substrate 1 through the half mirror 17. The irradiation intensity is, for example, 1 mW / mm ² . The structure 23 on the surface of the reaction substrate 1 irradiated with the LD light 16 emits fluorescence. This fluorescence is detected by the two-dimensional sensor camera 14 with the adjustment band-pass filter attached, and the Z-axis drive mechanism 8 is operated based on the obtained fluorescence image to adjust the focus. The adjustment method uses a known autofocus technique. For example, an image processing method or a phase difference detection method can be used. Next, the spot position of the reflected light of the LD light 16 is analyzed. The band pass filter for adjustment is removed, and the center coordinates are calculated by plotting the spot intensity of the LD reflected light in the X-axis direction and the Y-axis direction. When the LD light 16 is perpendicularly incident on the reaction substrate 1, the LD reflected light should be located at the center of the two-dimensional sensor camera 14, but when the reaction substrate 1 is tilted, the LD light 16 is It is not incident perpendicularly and is detected at a position shifted from the center of the two-dimensional sensor camera 14. When the spot of the LD reflected light is deviated from the center of the two-dimensional sensor camera 14, the α / β-axis drive mechanism 9 attached to the XY stage 7 is adjusted so that the spot center of the LD reflected light is the spot of the two-dimensional sensor camera 14. The inclination of the reaction substrate 1 is adjusted so as to be the center. After the adjustment, the irradiation of the LD light 16 is finished, and the half mirror 17 is removed. A band-pass filter for observation is attached, and irradiation with the laser beam 2a is started. Evanescent light is generated on the surface of the reaction substrate 1 by the laser light 2a totally reflected at the center of the observation field. The two-dimensional sensor camera 14 detects the fluorescence of the measurement sample 24 excited by the evanescent light. After the observation of the first visual field, the laser irradiation is terminated, and if necessary, the XY stage 7 is moved to move to the observation of the second visual field. After moving to the second field of view, the same operation as in the first field of view is performed again. The above operation is repeated to observe the entire observation field of the reaction substrate.

  A specific sequence analysis method is performed according to the following procedure. A single-stranded nucleic acid as a measurement sample is hybridized to a primer fixed to the structure 23 on the reaction substrate 1. The reaction is carried out, for example, at 60 ° C. for 10 minutes. The primer sequence only needs to be at least partially complementary to the single-stranded nucleic acid. The base length of the primer is preferably 10 or more in view of the hybridization efficiency.

Next, a base extension reaction is performed while sequentially adding labeled dATP, dTTP, dCTP, and dGTP, and the base sequence downstream of the primer is decoded. For example, first, a reaction solution containing Cy3-dATP and a DNA polymerase (20 nM Cy3-dATP, 0.1 U / μL Taq DNA polymerase, 10 mM Tris-HCl pH 7.8, ₂ mM MgCl ₂ ) Allow to react for minutes. Next, unreacted Cy3-dATP is removed with a washing buffer (10 mM Tris-HCl pH 7.8, ₂ mM MgCl ₂ ). Various reagents and buffers are stored in the reagent storage unit 15. The reagent storage unit 15 includes a sample solution container 15a, four types of labeled dNTP solution containers 15b, 15c, 15d, 15e, a dNTP mixture solution container 15f, a polymerase solution container 15g, a washing buffer container 15h, and the like.

A YAG laser having a wavelength of 532 nm is irradiated as excitation light, and fluorescence is generated from a fragment in which Cy3-dATP has been incorporated. In addition, since the fluorescence generated from the Cy3 molecule is quenched by irradiation with strong excitation light, it is desirable to observe with lower excitation light intensity during observation. The excitation light intensity is, for example, 1000 mW / mm ² . Addition of an oxygen scavenger is also effective as a means for suppressing quenching. This is because the quenching of the Cy3 molecule is caused by a reaction with dissolved oxygen in the solution. As the oxygen scavenger, for example, peroxidase or superoxide dismutase can be used. Based on the fluorescence observation data, the position of the fragment into which Cy3-dATP has been incorporated is specified, and the sequence is determined. After observing Cy3 fluorescence, strong excitation light is irradiated so that the already-derived fluorescence derived from Cy3-dATP does not occur in the next step. The above series of reaction steps are performed in the order of dATP, dTTP, dCTP, and dGTP. By performing this operation for 80 cycles, about 20 to 30 bases on the solid phase side of each fragment can be decoded. In this embodiment, Cy3 is used as the fluorescent dye, and a YAG laser having a wavelength of 532 nm is used as the excitation light source. However, the combination of label molecules and light sources is not limited to this.

In this embodiment, one type of fluorescent dye is used and base sequence decoding is performed by sequential reaction. However, another method may be used. For example, when using a labeled dNTP in which four different fluorescent dyes are bonded to the 3′-OH group of four types of dNTPs via a nitrobenzyl group, the dNTPs are reacted one by one as in this example. It is not necessary. That is, the fluorescent dye having a 3′-OH group serves as a protecting group, and the subsequent extension reaction does not proceed at the stage of incorporation. Using this fact, the base sequence is decoded. For example, four types of fluorescently labeled dNTPs and a reaction solution containing DNA polymerase (20 nM fluorescently labeled dNTP mixture, 0.1 U / μL Taq DNA polymerase, 10 mM Tris-HCl pH 7.8, ₂ mM MgCl ₂ ) React with fragment for 5 minutes. Next, unreacted fluorescently labeled dNTPs are removed with a washing buffer (10 mM Tris-HCl pH 7.8, ₂ mM MgCl ₂ ). The four types of fluorescence are observed by irradiating with excitation light, the fragment in which each fluorescently labeled dNTP is incorporated is identified, and the sequence is determined. As the four types of fluorescent dyes, for example, Cy3, Cy5, Cy5.5, Alexa fluor (registered trademark) 488 can be used. In order to prevent cross-contamination of fluorescence, it is preferable to select one having a fluorescence wavelength band as far as possible. As the excitation light, an excitation light source suitable for the wavelength characteristics of each fluorescent dye is used, or a target wavelength component is separated from multi-wavelength light using a band pass filter or the like. Similarly, fluorescence is observed by separating the target wavelength component using a bandpass filter suitable for the wavelength characteristics of each fluorescent dye. After observation, the fluorescent dye is separated by chemical or physical means, and the 3′-OH group of the fluorescently labeled dNTP incorporated immediately before is released. The means for separating is, for example, UV irradiation with a wavelength of 360 nm or less. The detached fluorescent dye is removed with a washing buffer. By performing the above series of operations a plurality of times, the base sequence can be decoded.

  With the above operation, the focus of the objective lens and the horizontal shift of the reaction substrate caused by the stage movement are automatically corrected and adjusted for the apparatus that performs fluorescence observation using evanescent light.

  According to the present embodiment, manual adjustment operation is unnecessary, and operability is improved as compared with the conventional method. In addition, since the reaction substrate can be kept horizontal at all times, the incident angle of the excitation light is constant, so that the penetration depth of the evanescent light is constant, which is more stable and reliable than the conventional method. improves. Further, since the irradiation area of the evanescent light can be always present at the center of the visual field, there is no loss and the throughput is improved as compared with the conventional method.

  In the first embodiment, an adjustment light source is prepared separately from the excitation light source, but these may be the same light source. Specifically, the same effects as those of the first embodiment are obtained by extracting the wavelength components for excitation and adjustment from the light source having multiple wavelength components. FIG. 4 shows an apparatus configuration of the present embodiment. The light emitted from the light source 25 is wavelength-separated by the dichroic mirror 26. The light source is 25, such as a xenon lamp. Since the wavelength component after separation is broad, it is made a single wavelength component by the bandpass filters 27 and 28 and the like. The surface of the reaction substrate is irradiated by an optical element 29 such as a mirror. Then, adjustment and observation are performed by the same procedure as in Example 1.

  In Example 1, although the wavelengths of the adjustment light source and the excitation light source are different, it is also possible to use light sources having the same wavelength. Specifically, the laser beam reflected by the reaction substrate is detected by a sensor, and the spot position of the reflected light is adjusted so as to be kept constant. Detect and correct to keep the relative positional relationship between the sample surface and the objective lens constant.

FIG. 5 shows an apparatus configuration of the present embodiment. FIG. 6 is a flowchart of fluorescence observation in this example. A specific method is performed according to the following procedure. After fixing the reaction substrate 1 to the XY stage 7, the first visual field is irradiated with the laser beam 2a with the irradiation intensity reduced. With the bandpass filter for fluorescence measurement removed, the structure 23 on the surface of the reaction substrate 1 is observed with scattered light. Based on the obtained scattered light image, the Z-axis drive mechanism 8 is driven to adjust the focus. The adjustment method uses a known autofocus technique. For example, an image processing method or a phase difference detection method can be used. For example, when the reaction substrate 1 is synthetic quartz and the medium is water, the incident angle θ is about 66 degrees. The irradiation intensity needs to be weak enough that the measurement sample 24 on the surface of the reaction substrate 1 is not quenched. The irradiation intensity is 1 mW / mm ² , for example. Next, the spot position of the evanescent light in the observation visual field is analyzed. The center coordinates are calculated by plotting the spot intensity of the evanescent light in the X-axis direction and the Y-axis direction. The irradiation position of the evanescent light is adjusted using the deflecting element 5 so that the center coordinates are the center of the observation field. The spot position of the reflected light is detected by the optical sensor 30, and the position information is stored as an initial state. After the adjustment is completed, fluorescence observation is performed. A band-pass filter for fluorescence observation is attached, and the laser beam 2a is irradiated with a normal irradiation intensity. The irradiation intensity is, for example, 1000 mW / mm ² . The two-dimensional sensor camera 14 detects the fluorescence of the measurement sample 24 excited by the evanescent light generated on the surface of the reaction substrate 1. After the observation of the first visual field, the laser irradiation is terminated, and if necessary, the XY stage 7 is moved to move to the observation of the second visual field. After moving to the second field of view, the Z-axis adjustment is performed again with the weak laser beam, as in the first field of view. Thereafter, the spot position of the reflected light is detected by the optical sensor 30 and compared with the initial state. If there is a deviation as a result of the comparison, the α / β-axis drive mechanism 9 is adjusted and corrected. After the adjustment, fluorescence observation is performed in the same manner as the first visual field observation procedure. The above operation is repeated to observe the entire observation field of the reaction substrate.

  In this embodiment, various adjustments are performed in the observation visual field. For example, by adjusting in the region adjacent to the observation visual field, the fear of quenching the measurement sample accompanying the adjustment can be eliminated.

  In the third embodiment, the tilt of the substrate is corrected by detecting the reflected light, but it is possible to correct without using the reflected light. Specifically, the tilt of the substrate is corrected based on the aspect ratio of the evanescent light spot.

FIG. 7 shows the apparatus configuration of this embodiment. FIG. 8 is a flowchart of fluorescence observation of this example. A specific method is performed according to the following procedure. After fixing the reaction substrate 1 to the XY stage 7, the first visual field is irradiated with the laser beam 2a with the irradiation intensity reduced. With the bandpass filter for fluorescence measurement removed, the structure 23 on the surface of the reaction substrate 1 is observed with scattered light. Based on the obtained scattered light image, the Z-axis drive mechanism 8 is driven to adjust the focus. The adjustment method uses a known autofocus technique. For example, an image processing method or a phase difference detection method can be used. For example, when the reaction substrate 1 is synthetic quartz and the medium is water, the incident angle θ is about 66 degrees. The irradiation intensity needs to be weak enough that the measurement sample 24 on the surface of the reaction substrate 1 is not quenched. The irradiation intensity is 1 mW / mm ² , for example. Next, the spot position of the evanescent light in the observation visual field is analyzed. The center coordinates are calculated by plotting the spot intensity of the evanescent light in the X-axis direction and the Y-axis direction. The irradiation position of the evanescent light is adjusted using the deflecting element 5 so that the center coordinates are the center of the observation field. Since the shape of the evanescent irradiation region depends on the beam shape of the laser light 2a, it is circular or elliptical. The aspect ratio of the evanescent light irradiation region is obtained from the intensity plot used in the above-described calculation of the center coordinates, and the information is stored as an initial state. After the adjustment is completed, fluorescence observation is performed. A band-pass filter for fluorescence observation is attached, and the laser beam 2a is irradiated with a normal irradiation intensity. The irradiation intensity is, for example, 1000 mW / mm ² . The two-dimensional sensor camera 14 detects the fluorescence of the measurement sample 24 excited by the evanescent light generated on the surface of the reaction substrate 1. After the observation of the first visual field, the laser irradiation is terminated, and if necessary, the XY stage 7 is moved to move to the observation of the second visual field. After moving to the second field of view, the Z-axis adjustment is performed again with the weak laser beam, as in the first field of view. Thereafter, the aspect ratio of the evanescent light irradiation region is calculated and compared with the initial state. If there is a deviation as a result of the comparison, the α / β-axis drive mechanism 9 is adjusted and corrected. After the adjustment, fluorescence observation is performed in the same manner as the first visual field observation procedure. The above operation is repeated to observe the entire observation field of the reaction substrate.

  In this embodiment, as in the third embodiment, for example, by performing adjustment in a region adjacent to the observation visual field, it is possible to eliminate the fear of quenching the measurement sample accompanying the adjustment.

  The present invention is useful for single molecule fluorescence observation. Therefore, it can be widely used in the life science field including biology, chemistry, and medicine.

DESCRIPTION OF SYMBOLS 1 Reaction board | substrate 2 Laser apparatus 2a Laser beam 3 (lambda) / 4 wavelength plate 4 Lens 5 Deflection element 6 Prism 7 Stage 8 Z-axis drive mechanism 9 alpha / beta-axis drive mechanism 10 Light emission 11 Objective lens 12 Filter unit 13 Imaging lens 14 2 Dimensional sensor camera 15 Reagent storage unit 15a Sample solution container 15b, 15c, 15d, 15e Labeled dNTP solution container 15f dNTP mixture solution container 15g Polymerase solution container 15h Wash buffer container 16 LD light 17 Half mirror 18 Light source for adjustment (LD)
19 Waste liquid container 20 Control PC
21 Monitor 22 Transparent member 23 Structure 24 Measurement sample 25 Light source 26 Dichroic mirror 27 Band pass filter 1
28 Band pass filter 2
29 Mirror 30 Optical sensor 31 Dispensing unit 32 Liquid feeding tube 33 Waste liquid tube

Claims (1)

In a fluorescence observation apparatus using evanescent light, a method for correcting a positional deviation of a substrate accompanying a stage movement,
Irradiate the substrate with excitation light,
Detecting an evanescent light irradiation area generated on the surface of the substrate;
Obtain the aspect ratio of the evanescent light irradiation area,
The aspect ratio is compared with the aspect ratio of the initial state obtained in advance,
A method of adjusting the inclination of the substrate so as to correct a deviation between the two.

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